The present disclosure generally relates to the field of imaging and to microscopy systems and methods. More particularly, and without limitation, the disclosed embodiments relate to systems and methods for volumetric imaging through the use of axial chromatic aberrations in an optical system.
Fluorescence microscopy uses principles of fluorescence to highlight structures for examination instead of light absorption, phase or interference effects. It can be used, for example, in biology and other disciplines for characterizing samples. Fluorescence microscopy is characterized by using incident light instead of the transmitted light employed in conventional brightfield transmitted-light microscopy. In the latter approach, light is shone on the sample and passes through it, and the effects of absorption create the image. In fluorescence microscopy, fluorophores in the sample are activated via an excitation light beam directed via the objective lens. The fluorophores emit a fluorescent light that creates the image.
When imaging thick samples, techniques can be employed to obtain images of high resolution in the axial direction. One such approach is confocal microscopy. The most common of these approaches is to use one confocal pinhole, or for higher throughput, an array of such pinholes, that rejects light emitted from planes that are outside of the focal plane. By blocking light from outside the focal plane, confocal microscopes achieve good axial resolution because the haze of out-of-focus objects is eliminated. In confocal microscopy, the excitation light source, usually a laser beam, is reflected by a dichroic filter or mirror. In scanning systems, scanning mirrors raster the beam across the sample or the sample is scanned in the transverse direction with the laser spot being fixed. The sample fluorescence light then passes back through the objective and is descanned. Thereafter, the light passes through the dichroic filter and pinhole to a photomultiplier tube detector.
A two dimensional image is generated by translating the pinholes or sample laterally. To generate a virtual volumetric image, the sample or objective is translated axially and the pinholes (or sample) are (is transversally) scanned to build up an image in that plane. This results in a significant reduction in volumetric imaging rates.
Fluorescence light sheet imaging is a technique that exhibits volumetric imaging capabilities. In this approach, a sheet of light at the excitation wavelength is sent in a plane that is perpendicular to the axis of the microscope objective lens that is collecting the light emissions. Out-of-focus light is not generated by ensuring that the thickness of the light sheet is suitably small. In this way, an entire cross-sectional plane of a sample can be captured at one time. The light sheet can be translated up and down to capture different planes.
However, conventional light sheet microscope setups have several drawbacks. For example, they are very particular, requiring access to the side of the sample as well as requiring multiple microscope objective lenses to be brought near the sample. Light sheet microscopy is, therefore, not suitable for imaging microscope slides, for example. Furthermore, the field of view of light sheet microscopes is limited.